Matrix metalloproteinases (MMPs) play a major role in the turnover of extracellular matrix (ECM) during cancer invasion and metastasis, and tissue inhibitors of metalloproteinases (TIMPs) control MMPs, thus maintaining a balanced ECM catabolism under physiological conditions. The aim of this study was to assess the behavior of some MMPs (FASEB J., 7, 1993, 1434; Cancer Metastasis Rev., 9(4) 1990, 289) and TIMPs (Biochem. Biophys. Res. Commun., 301, 2003, 1069; FASEB J., 7, 1993, 1434; Nature, 370, 1994, 61). Competitive RT-PCR, gelatin-substrate zymography, and ELISA techniques were used for quantification. The hepatitis B virus (HBV)-DNA-containing hepatocellular carcinoma cell lines, Hep3B, HepG2-HBV and HFF-T2 contain highly activated matrix metallproteinase-9 (MMP-9), which is rarely found in normal liver cell lines such as the Chang lines. MMP-9 activities of HFH-T2, HepG2-HBV and Hep3B were significantly higher than that of non-HBV-hepatocellular carcinoma SK-Hep1 and HepG2 (HCC origin, HBV not detected), as assayed by gelatin zymography. Low levels of TIMP-1 and TIMP-3 were observed in HFH-T2, HepG2-HBV and Hep3B, while the TIMP-2 level was high, as evidenced by reverse zymography. In contrast, 3 TIMP-1, -2 and -3 were largely detected in Chang, HepG2 and SK-Hep1 cells. To investigate the nature of the quantitative regulation of MMPs and TIMPs for these cell lines at the transcriptional levels, a reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out. Not only MMP-9 mRNAs of HFH-T2, HepG2-HBV and Hep3B but also MMP-9 mRNA of SK-Hep1 and HepG2 were highly expressed with no major differences among these four cell lines. However, TIMP-1 and TIMP-3 mRNAs of HFH-T2, HepG2-HBV and Hep3B were markedly reduced, while those of SK-Hep1, HepG2 and Chang cells were maintained at high levels. Finally, an invasion assay using matrigel indicated in an increase in invasiveness in HFH-T2, HepG2-HBV and Hep3B cells, but a decrease in invasiveness of Chang, HepG2 and SK-Hep1 cells. These results indicate that the overexpression of MMP-9 mRNAs and the suppression of TIMP-1 and TIMP-3 in HFH-T2, HepG2-HBV and Hep3B were the result of HBV transfection. Based on these results, it is concluded that HBV affects the malignance of hepatocellular cancer by elevating MMP-9 activity, and suppressing TIMP-1 and TIMP-3.